WO2021241802A1 - Agent arni ciblant le facteur de croissance des tissus conjonctifs et son utilisation - Google Patents

Agent arni ciblant le facteur de croissance des tissus conjonctifs et son utilisation Download PDF

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WO2021241802A1
WO2021241802A1 PCT/KR2020/010781 KR2020010781W WO2021241802A1 WO 2021241802 A1 WO2021241802 A1 WO 2021241802A1 KR 2020010781 W KR2020010781 W KR 2020010781W WO 2021241802 A1 WO2021241802 A1 WO 2021241802A1
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nucleic acid
acid molecule
rnai
sense strand
inducing
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홍선우
박준현
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Olix Pharmaceuticals Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • the present application relates to a nucleic acid molecule for preventing or treating a disease through the phenomenon of RNA interference and a use thereof.
  • Age-related macular degeneration is the leading cause of blindness worldwide.
  • Subretinal fibrosis is a phenomenon in which fibrous tissue proliferates around the retina and induces macular edema and detachment to permanently damage vision.
  • Major chronic retinal diseases such as wet age-related macular degeneration, proliferative diabetic retinopathy, proliferative vitreoretinopathy, central retinal vein occlusion, and optic neuropathy Retinal fibrosis is common in most cases, but there is no cure.
  • CTGF connective tissue growth factor
  • the present inventors have developed an RNA preparation using RNA interference technology as a result of earnest research efforts to develop a new safe drug that can treat wet macular degeneration and/or subretinal fibrosis patients.
  • Another object of the present invention is to provide an RNAi agent for the treatment of ocular diseases, in particular subretinal fibrosis and/or wet age-related macular degeneration, and a pharmaceutical use thereof.
  • one aspect provides a nucleic acid molecule for inducing double-stranded RNAi targeting CTGF.
  • Another aspect provides a pharmaceutical composition for preventing or treating wet macular degeneration and/or subretinal fibrosis comprising the RNAi-inducing nucleic acid molecule as an active ingredient.
  • the nucleic acid molecule is a chronic retinal disease, such as wet age-related macular degeneration, proliferative diabetic retinopathy, proliferative vitreoretinopathy, central retinal vascular occlusion, optic neuropathy, and/or subretinal fibrosis and/or choroidal neovascularization ( Choroidal Neovascularization, CNV) can be utilized as an active ingredient of a pharmaceutical composition for inhibiting the formation or progression.
  • a chronic retinal disease such as wet age-related macular degeneration, proliferative diabetic retinopathy, proliferative vitreoretinopathy, central retinal vascular occlusion, optic neuropathy, and/or subretinal fibrosis and/or choroidal neovascularization ( Choroidal Neovascularization, CNV) can be utilized as an active ingredient of a pharmaceutical composition for inhibiting the formation or progression.
  • FIG. 1 shows the change in CTGF mRNA level according to the treatment of siRNA according to the present invention.
  • 2a to 2d show changes in the level of CTGF protein according to the treatment of siRNA according to the present invention.
  • 3a and 3b are results of confirming the change in CNV volume after intraocular injection of siRNA according to the present invention into a CNV monkey model.
  • 4a to 4b are results of confirming the therapeutic efficacy of the siRNA according to the present invention in a mouse model of subretinal fibrosis following CNV induction;
  • Figure 4a shows collagen deposition changes in CNV lesions
  • Figure 4b shows CTGF immunoreactivity in CNV lesions.
  • 4c to 4e are results of confirming the therapeutic efficacy of the siRNA according to the present invention in a mouse model of subretinal fibrosis caused by retinal detachment,
  • Figure 4c shows changes in the subretinal fibrosis area
  • Figure 4d shows the change of the collagen deposition area
  • Figure 4e shows changes in CTGF protein levels.
  • 5a to 5f are results of confirming the therapeutic efficacy of the siRNA according to the present invention in the NHP CNV monkey model
  • 5A to 5D show a change in leakage area, a change in retinal thickness, a change in collagen deposition area, and a change in CTGF protein level, respectively, according to OLX201A-093-8 administration.
  • 5E and 5F show the change in leakage area and the change in retinal thickness according to the administration of OLX201A-093-39, respectively.
  • One aspect is a nucleic acid molecule for inducing double-stranded RNAi comprising a sense strand and an antisense strand,
  • the sense strand has a length of 15 to 17 nt, and at least 15 contiguous nucleotides in the sense strand are complementary to the antisense strand;
  • the sense strand comprises one or more chemical modifications
  • the antisense strand (5'->3') comprises the sequence of P-mAAAUCUmGmGCUUmGUUmA*C*mA*mG*mG,
  • * is a phosphorothioate bond
  • m is 2'-O- methyl substitution
  • P is a 5'-phosphate group binding
  • Another aspect is a nucleic acid molecule for inducing double-stranded RNAi comprising a sense strand and an antisense strand,
  • the antisense strand has a length of 19 to 21 nt, and at least 15 contiguous nucleotides in the antisense strand are complementary to the sense strand;
  • the antisense strand comprises at least one or more chemical modifications
  • the sense strand (5'->3') comprises the sequence of mGmUmAACAAGCCAGAU*U*U*Lp,
  • * is a modification with a phosphorothioate bond
  • m is a substitution with 2'-O-methyl
  • Lp is a lipophilic moiety, providing a nucleic acid molecule for inducing RNAi.
  • nucleic acid molecule for inducing RNAi comprising double-stranded siRNA, which inhibits the expression of CTGF
  • the double-stranded siRNA includes an antisense strand having the nucleotide sequence of SEQ ID NO: 1 (5'-AAAUCUGGCUUGUUACAGG-3') and a sense strand having the nucleotide sequence of SEQ ID NO: 2 (5'-GUAACAAGCCAGAUUU-3'), ,
  • the antisense strand and the sense strand are complementary to each other, so that the 5' end of the antisense strand and the 3' end of the sense strand form a blunt end,
  • RNAi induction A nucleic acid molecule for use is provided.
  • RNA interference generally refers to the art of inhibiting or down-regulating gene expression in a cell by causing the destruction of a specific target RNA and mediated by sequence-specific nucleic acid molecules. refers to a commonly known biological process in Also, the term RNAi may be equivalent to other terms used to describe sequence specific RNA interference, such as post-transcriptional gene silencing, translational repression, transcriptional repression, or epigenetics.
  • the siRNA molecule can be used to silence a gene at the post-transcriptional level or the pre-transcriptional level.
  • modulation of gene expression by an siRNA molecule may result from siRNA-mediated cleavage of mRNA through RISC.
  • nucleic acid molecule for inducing RNAi refers to gene expression or virus by mediating the RNA interference in a sequence-specific manner.
  • the term may refer to both an individual nucleic acid molecule, a plurality of said nucleic acid molecules, or a pool of said nucleic acid molecules.
  • the siRNA may be an asymmetric double-stranded nucleic acid molecule comprising self-complementary sense and antisense strands.
  • the term "gene” should be considered in its broadest sense, and may encode a structural protein or a regulatory protein.
  • the regulatory protein includes a transcription factor, heat shock protein, or a protein involved in DNA/RNA replication, transcription and/or translation.
  • the target gene to be suppressed is inherent in the viral genome, and may be integrated into an animal gene or exist as an extrachromosomal component.
  • the term “antisense strand” refers to a generally accepted meaning in the art.
  • the term may refer to the nucleotide sequence of an siRNA molecule having complementarity to CTGF RNA.
  • the antisense strand of the siRNA molecule may include a nucleic acid sequence having complementarity to the sense strand of the siRNA molecule.
  • the antisense strand of the siRNA molecule may be referred to as an antisense region or a guide strand.
  • the term “sense strand” refers to a generally accepted meaning in the art. In the context of the siRNA molecules described herein, the term may refer to the nucleotide sequence of the siRNA molecule, which has complementarity to the antisense strand of the siRNA molecule. In addition, the sense strand of the siRNA molecule may comprise a nucleic acid sequence having homology or sequence identity to the target nucleic acid sequence. Also, in one embodiment, the sense strand of the siRNA molecule may be referred to as a sense region or a passenger strand.
  • complementarity refers to a generally accepted meaning in the art.
  • the term generally refers to the formation or presence of hydrogen bond(s) between one nucleic acid sequence and another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional types of bonding described herein. can refer to Perfect complementarity may mean that all contiguous residues of a nucleic acid sequence hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • Partial complementarity within a nucleic acid molecule can occur with various mismatch or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches, non-nucleotide linkers, or non-base pair nucleotides).
  • the partial complementarity can be expressed by bulges, loops ( loops), overhangs, or blunt ends.
  • blunt end refers to the generally accepted meaning in the art.
  • the term may refer to the terminus of a double-stranded siRNA molecule that lacks overhanging nucleotides.
  • the siRNA molecule described herein may be such that the 5'-end of the antisense strand and the 3'-end of the sense strand form a blunt end.
  • CTGF Connective Tissue Growth Factor
  • CCN2 connective tissue growth factor
  • CTGF is involved in biological activities including cell adhesion, migration, proliferation, angiogenesis, skeletal development, and repair of tissue spheroids. It is known that, in particular, it is known to be closely related to fibrotic disease.
  • the CTGF protein can be interpreted as including naturally occurring wild-type CTGF and functional variants thereof, and the sequence of the CTGF protein or a gene encoding the same can be obtained from a known database such as GenBank of the National Institutes of Health of the United States of America.
  • expression refers to the generally accepted meaning in the art.
  • the term can generally refer to the process by which a gene ultimately produces a protein.
  • Such expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, or translation.
  • expression levels can be determined or monitored by detection of mRNA levels or protein levels.
  • inhibitors or reduction as used in reference to CTGF gene expression in a subject refer to a statistically significant decrease compared to an untreated group or a normal control group.
  • the reduction is, for example, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. It may be above, but it may be below the detection level, depending on the detection or measurement method.
  • siRNA is a short interfering RNA (RNA) and is involved in RNAi (RNA interference) action.
  • RNAi is an intracellular gene regulation mechanism first discovered in Caenorthabditis elegans in 1998. The mechanism of action is known to induce target gene degradation by complementary binding of the antisense strand among the RNA double strands injected into the cell to the mRNA of the target gene. It is a promising new drug development candidate technology.
  • siRNA is an effective method for directly regulating the expression of a target gene, it is difficult to develop a therapeutic agent due to these problems.
  • asymmetric shorter duplex siRNA (asiRNA) is an asymmetric RNAi-inducing structure having a shorter double helix length compared to the 19+2 structure of conventional siRNA. It is a technology that overcomes problems such as the off-target effect, saturation of RNAi mechanism, and immune response by TLR3 confirmed in existing siRNA structure technology, and thus it is possible to develop new RNAi drugs with low side effects.
  • an asymmetric siRNA comprising a sense strand and an antisense strand complementary to the sense strand is presented in this embodiment, and the siRNA according to an embodiment does not cause problems such as off-target effect and saturation of RNAi mechanism. While stably maintaining high transduction efficiency, it is possible to effectively suppress the expression of the CTGF gene to a desired degree.
  • an asymmetric siRNA (asymmetric siRNA: asiRNA) targeting CTGF was designed and manufactured, and after transfection of the asiRNA into cells expressing CTGF, a nucleic acid molecule for inducing RNAi with excellent knockdown efficiency, that is, CTGF asiRNA was selected.
  • the nucleic acid molecule for inducing RNAi includes an antisense strand of SEQ ID NO: 1 and a sense strand of SEQ ID NO: 2, and each strand may have a chemical modification introduced thereinto.
  • the 5'-end of the antisense strand and the 3'-end of the sense strand may form a blunt end.
  • the sense strand or the antisense strand may include one or more chemical modifications.
  • siRNA cannot pass through the cell membrane for reasons such as high negative charge and high molecular weight due to the phosphate backbone structure, and it is rapidly degraded and removed from the blood, so it is difficult to deliver a sufficient amount for RNAi induction to the actual target site.
  • in vitro delivery many high-efficiency delivery methods using cationic lipids and cationic polymers have been developed, but in the case of in vivo, it is difficult to deliver siRNA as high as in vitro, and it is There is a problem in that the siRNA delivery efficiency is reduced due to the interaction.
  • a nucleic acid molecule for inducing RNAi having cell-penetrating ability by introducing a chemical modification into the asymmetric siRNA structure is presented, and more specifically, effective and intracellular delivery without a separate carrier.
  • An asymmetric siRNA construct (cell-penetrating asymmetric siRNA) is presented.
  • (1) introduction of a lipophilic moiety at the 3' end of the sense strand can allow siRNA to easily penetrate the cell membrane, and (2) phosphorothioate the phosphate backbone adjacent to the end of the sense strand or antisense strand. Substitution with eg, confer resistance to exohydrolase, and may enable uptake into cells and bioavailability of siRNA in vivo. (3) Substitution of methyl, methoxy, etc. for the -OH group at the 2' carbon position of the sugar structure can confer resistance to nucleolytic enzymes, lower siRNA immunogenicity, and reduce off-target effects. , (4) The substitution of fluoro for the -OH group at the 2' carbon position of the sugar structure imparts stability to the double strand, increases stability in serum, and enables efficient silencing in vitro and in vivo.
  • the antisense strand comprises the sequence P-mAAAUCUmGmGCUUmGUUmA*C*mA*mG*mG, wherein * is a modification with a phosphorothioate bond, m is a substitution with 2'-0-methyl, and P denotes a bond with a 5'-phosphate group.
  • the sense strand has a length of 15 to 17 nt, and at least 15 contiguous nucleotides in the sense strand are complementary to the antisense strand;
  • the sense strand may include at least one or more chemical modifications.
  • the sense strand comprises the sequence mGmUmAACAAGCCAGAU*U*U*Lp, wherein * is a modification with a phosphorothioate bond, m is a substitution with 2'-0-methyl, and Lp is a 3'-lipophilic moiety It means the introduction of tea.
  • the lipophilic moiety includes cholesterol, tocopherol, stearic acid, retinoic acid, docosahexaenoica cid (DHA), palmitic acid, linoleic acid, linolenic acid, and It may be selected from long-chain fatty acids having 10 or more carbon atoms, preferably cholesterol, DHA, or palmitic acid. Most preferred is palmitic acid.
  • the sense strand comprises any one sense strand selected from '-Cholesterol binding, DHA means 3'-Docosahexaenoic acid (DHA) binding, and PA means 3'-Palmitic acid binding:
  • Another aspect provides a pharmaceutical composition for preventing or treating an ocular disease comprising a nucleic acid molecule for inducing RNAi as an active ingredient.
  • the pharmaceutical composition contains or uses the above-described nucleic acid molecule for inducing RNAi as it is, descriptions of common content between the two are omitted in order to avoid excessive complexity of the present specification.
  • the pharmaceutical composition Since the pharmaceutical composition has a function of inhibiting abnormal angiogenesis by inhibiting expression of the CTGF gene, it can be utilized as an active ingredient of a pharmaceutical composition for the prevention or treatment of ocular diseases accompanying vascular abnormalities.
  • the ocular disease may be, for example, subretinal fibrosis or subretinal fibrosis resulting from wet age-related macular degeneration, proliferative diabetic retinopathy, proliferative vitreous retinopathy, central retinal vascular occlusion, optic nerve neuropathy, wherein "Macular degeneration” is an eye disease with symptoms that affect vision due to abnormal growth of new blood vessels and damage to the macula. Macular degeneration mainly occurs in the age group over 50, and is divided into dry macular degeneration and wet macular degeneration.
  • the term "effective ingredient” means an appropriate effective amount of an ingredient that affects a beneficial or desirable clinical or biochemical outcome. Specifically, it may refer to an effective amount of an agent, active agent, or nucleic acid molecule.
  • Said effective amount may be administered one or more times and may be administered to prevent a disease, or to treat a disease state, including but not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., not worsening) of the disease state, delaying disease progression, or It may be an amount suitable for reducing the rate, or for amelioration or temporary alleviation and alleviation (partial or total) of the disease state.
  • prevention refers to any action that blocks the occurrence of a disease in advance, suppresses the disease, or delays the progression. For example, it refers to preventing, preventing, or defending or protecting from the occurrence of, said ocular disease or characteristic characteristic thereof.
  • treatment refers to both therapeutic treatment and prophylactic or prophylactic measures.
  • it refers to any action that improves or beneficially changes the symptoms of the disease. For example, to prevent, reduce or ameliorate the ocular disease or characteristic characteristic thereof, or to delay (attenuate) the progression of the ocular disease or characteristic characteristic thereof in a subject.
  • the term “effective amount” refers to a generally accepted meaning in the art.
  • the term generally refers to a molecule, compound, or construct that will elicit an intended biological response (eg, a beneficial response) in a cell, tissue, system, animal, or human being sought by a researcher, veterinarian, physician, or other clinician, etc. It can mean quantity.
  • a “therapeutically effective amount” refers to a desired medical response to the extent that, for example, there is a therapeutically relevant change in a measurable parameter associated with a disease or disorder, such that a particular clinical treatment can be considered effective. It can refer to an amount of a molecule, compound, or construct that can be derived.
  • a therapeutically effective amount of a drug for the treatment of said disease or disorder may be that amount necessary to effect a therapeutically relevant change in said parameter.
  • a pharmaceutical composition comprising a nucleic acid molecule according to the present invention may be administered intraocularly.
  • Intraocular administration of the nucleic acid molecule may be by injection or direct (eg, topical) administration to the eye, so long as the route of administration allows the nucleic acid molecule to enter the eye.
  • suitable intraocular routes of administration include intravitreal, intraretinal, subretinal, subtenon, peri-orbital and retro-orbital, Including intraconjunctival, subconjunctival, trans-corneal and trans-scleral administration.
  • Another aspect provides a method of treating an ocular disease comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition.
  • the method for treating an ocular disease includes or uses the above-described RNAi-inducing nucleic acid molecule or pharmaceutical composition as it is, descriptions of common contents therebetween are omitted in order to avoid excessive complexity of the present specification.
  • the term "subject” refers to a subject in need of treatment for a disease, specifically an ocular disease, and more specifically, a human or non-human primate, mouse, dog, cat, horse, It may include all mammals such as cattle, sheep, pigs, goats, camels, and antelopes.
  • siRNA targeting CTGF was synthesized, and various chemical modifications (2'OMe, PS, Fluoro) and siRNA introduced with lipophilic moieties such as cholesterol, DHA, and PA at the 3' end of the sense strand were used.
  • 2'OMe, PS, Fluoro siRNA introduced with lipophilic moieties such as cholesterol, DHA, and PA at the 3' end of the sense strand were used.
  • lipophilic moieties such as cholesterol, DHA, and PA at the 3' end of the sense strand were used.
  • Table 1 which methods are well known in the art.
  • cholesterol-TEG-CPG manufactured by LGC Prime Synthesis
  • those disclosed in Table 3 were used for the synthesis of siRNA into which cholesterol, DHA and PA, which are lipophilic moieties, were introduced.
  • A549 cells were treated with 6, 16, 31, 63, 125, 250, 500, and 1000 nM of siRNA, respectively, and incubated (free uptake), and then the expression level of CTGF mRNA was measured by realtime qPCR. Specifically, A549 cells were seeded in a 24-well plate at 3 x 10 4 cells/well, and 24 hours later, 100 nM of cp-asiRNA was added thereto, and then the cells were incubated in Opti-MEM media conditions. did.
  • CTGF protein following administration of OLX201A-093-8 in retinal pigmented epithelium (RPE) cells of 9-week-old male C57BL/6 mice was confirmed by western blot analysis. Specifically, 0.8 ⁇ l of a solution of 0.125, 0.25, and 0.5 ⁇ g of OLX201A-093-8 in 10 mM PBS was intravitreally injected into C57BL/6 mice (6 mice per group, 12 eyes) (intravitreal injection, IVT) ( Day 0).
  • RPE retinal pigmented epithelium
  • RPE was isolated from the mice, RIPA buffer (SIGMA, R0278) was added, and homogenized using a tissue grinder pestle (Scienceware, 199230001) and a sonicator (Sonics, VC505). Then, the protein was quantified from the supernatant obtained by centrifugation using the BCA Protein Assay Kit (Thermo, 23225), and 5 ⁇ g of the protein for each sample was treated with 8-16% Precast Gel (Bio-rad, 456-1106). was used for electrophoresis, and transferred to a PVDF membrane (Bio-rad, 1622017).
  • CTGF protein level was reduced as shown in Tables 9 and 10 and FIGS. 2c and 2d .
  • OLX201A-093-8, OLX201A-093-37, OLX201A-093-38 and OLX201A in 9-week-old male C57BL/6 mice induced by laser photocoagulation induced choroidal neovascularization (CNV).
  • CNV laser photocoagulation induced choroidal neovascularization
  • the treatment efficacy according to -093-39 administration was evaluated. Specifically, immediately after induction of laser photocoagulation (Power: 140 mW, Duration: 100 ms, Size: 75 ⁇ m, 4 lasers/eye) in mice (8 mice per group, 8 eyes), 0.25, 0.5 and 1 ⁇ g 0.8 ⁇ l of a solution of OLX201A-093-8 mixed with 1X PBS was intravitreally administered.
  • the RPE flat isolated from the mouse eye was stained with Isolectin B4 (Vector laboratories, FL-1201) specific for vascular endothelial cells. Then, using a confocal microscope (Leica, TCS SP8), images were taken from the beginning to the end point at which fluorescence was observed. The CNV volume was measured by quantifying the IB4 stained area from the image taken through Image J software, and the treatment efficacy was evaluated by comparing the % with the negative control group (1X PBS administration group).
  • Tables 11 to 14 below show the decrease in CNV volume according to the administration of each cp-siRNA prepared in Example 1. (See Fig. 3)
  • 37, OLX201A-093-38, and OLX201A-093-39 treatment according to the administration was evaluated.
  • choroidal neovascularization was achieved by laser photocoagulation (Power: 140 mW, Duration: 80 ms, Size: 75 ⁇ m, 11 lasers/eye) in 10-week-old male C57BL/6 mice (3 mice per group, 4 eyes). induced.
  • the retinal paraffin section prepared in the same manner as above was used with CTGF antibody (1:100; Abcam, Ab125943) and ABC HRP Kit (Vector laboratories, PK-6101). After that, the color was developed with DAB substrate Kit (Abcam, ab64238) and images were taken using a microscope (Nikon, ECLIPSE TS100). In the captured image, the CTGF protein expression level was evaluated by analyzing the region showing CTGF expression in the subretinal fibrosis region with Image J software, and as a result, it was confirmed that the CTGF protein was reduced. (See Table 16, Figure 4b)
  • CTGF protein expression level was evaluated by analyzing the region showing CTGF expression in the subretinal fibrosis region with Image J software. As a result, as a result of administration of OLX201A-093-8, the CTGF protein level was decreased. (See Table 19, Figure 4e)
  • ffERG full-field electroretinogram
  • siRNAs OLX201A-093-8, OLX201A-093-37, OLX201A synthesized in Example 1 were used in a rhesus monkey model in which choroidal neovascularization (CNV) was induced by laser photocoagulation. The therapeutic efficacy of -093-39 was evaluated.
  • CNV choroidal neovascularization
  • the therapeutic efficacy of OLX201A-093-8 was evaluated by analyzing the leakage area, retinal thickness, collagen deposion, and CTGF protein expression levels in the monkey CNV model of OLX201A-093-8. Specifically, laser photocoagulation (Energy: 650-700 mW, Laser wavelength: 532 nm, Diameter of facula: 50 ⁇ m, Exposure time: 0.1 sec, 8 spots/eye) was induced, and after 3 weeks, 50 ⁇ l of a solution of 0.05 and 0.1 mg of OLX201A-093-8 mixed with 1X PBS was intravitreally administered.
  • laser photocoagulation (Energy: 650-700 mW, Laser wavelength: 532 nm, Diameter of facula: 50 ⁇ m, Exposure time: 0.1 sec, 8 spots/eye) was induced, and after 3 weeks, 50 ⁇ l of a solution of 0.05 and 0.1 mg of OLX201A-093-8 mixed with 1X PBS was
  • FFA fundus fluorescein angiography
  • OCT optical coherence tomography
  • the therapeutic efficacy was evaluated by analyzing leakage area and retinal thickness in the monkey CNV model of OLX201A-093-39. Specifically, laser photocoagulation (Energy: 600-670 mW, Laser wavelength: 532 nm, Diameter of facula: 50 ⁇ m, Exposure time: 0.1 sec, 7-8 spots/eye) was induced, and after 3 weeks, 50 ⁇ l of a solution of 0.1 mg of OLX201A-093-39 mixed with 1X PBS was intravitreally administered.
  • laser photocoagulation Esnergy: 600-670 mW, Laser wavelength: 532 nm, Diameter of facula: 50 ⁇ m, Exposure time: 0.1 sec, 7-8 spots/eye
  • FFA fundus fluorescein angiography

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Abstract

La présente invention concerne un agent ARNi ciblant le facteur de croissance du tissu conjonctif et son utilisation, et fournit une molécule d'acide nucléique induisant l'ARNi pour inhiber l'expression du facteur de croissance du tissu conjonctif (CTGF), et une composition pharmaceutique pour la prévention ou le traitement de la fibrose sous-rétinienne et/ou de la dégénérescence maculaire humide liée à l'âge, la composition pharmaceutique comprenant la molécule d'acide nucléique induisant l'ARNi comme principe actif.
PCT/KR2020/010781 2020-05-26 2020-08-13 Agent arni ciblant le facteur de croissance des tissus conjonctifs et son utilisation Ceased WO2021241802A1 (fr)

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PCT/KR2020/010787 Ceased WO2021241803A1 (fr) 2020-05-26 2020-08-13 Agent arni ciblant le myd88 et son utilisation

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EP (1) EP4159858A4 (fr)
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KR (3) KR102421750B1 (fr)
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AU (1) AU2020450890B2 (fr)
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KR100949791B1 (ko) * 2007-12-18 2010-03-30 이동기 오프-타겟 효과를 최소화하고 ＲＮＡｉ 기구를 포화시키지않는 신규한 ｓｉＲＮＡ 구조 및 그 용도
WO2011119887A1 (fr) * 2010-03-24 2011-09-29 Rxi Pharmaceuticals Corporation Arn interférant dans des indications dermiques et fibrosiques
KR20180128423A (ko) * 2016-04-11 2018-12-03 올릭스 주식회사 연결 조직 성장 인자를 표적화하는 rna 복합체를 사용한 특발성 폐 섬유증의 치료 방법
KR20190037166A (ko) * 2017-09-28 2019-04-05 올릭스 주식회사 결합 조직 성장 인자를 표적으로 하는 rna 복합체를 함유하는 노인성 황반변성의 예방 또는 치료용 약학 조성물

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US20100035969A1 (en) * 2004-12-23 2010-02-11 Alcon, Inc. RNAi INHIBITION OF CTGF FOR TREATMENT OF OCULAR DISORDERS
KR100949791B1 (ko) * 2007-12-18 2010-03-30 이동기 오프-타겟 효과를 최소화하고 ＲＮＡｉ 기구를 포화시키지않는 신규한 ｓｉＲＮＡ 구조 및 그 용도
WO2011119887A1 (fr) * 2010-03-24 2011-09-29 Rxi Pharmaceuticals Corporation Arn interférant dans des indications dermiques et fibrosiques
KR20180128423A (ko) * 2016-04-11 2018-12-03 올릭스 주식회사 연결 조직 성장 인자를 표적화하는 rna 복합체를 사용한 특발성 폐 섬유증의 치료 방법
KR20190037166A (ko) * 2017-09-28 2019-04-05 올릭스 주식회사 결합 조직 성장 인자를 표적으로 하는 rna 복합체를 함유하는 노인성 황반변성의 예방 또는 치료용 약학 조성물

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KR20210146196A (ko) 2021-12-03
KR102643127B1 (ko) 2024-03-06
JP7539493B2 (ja) 2024-08-23
KR102421751B1 (ko) 2022-07-19
JP2023528593A (ja) 2023-07-05
KR20210146195A (ko) 2021-12-03
KR102421750B1 (ko) 2022-07-19
CA3179876A1 (fr) 2021-12-02
AU2020450890B2 (en) 2025-04-10
KR20220116378A (ko) 2022-08-22
US20240093188A1 (en) 2024-03-21
EP4159858A1 (fr) 2023-04-05
EP4159858A4 (fr) 2024-07-24
CN115698287A (zh) 2023-02-03
AU2020450890A1 (en) 2023-02-02

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